Download K916-100, Cell invasion Assay Kit (collagen I) 96-well 8 um

FOR RESEARCH USE ONLY!
EZCellTM Cell Invasion Assay Kit (Collagen I), 96-well, 8 µm
7/15
(Catalog # K916-100; 100 assays; Store at -20°C)
I.
Introduction:
Cell invasion is the ability of cells to migrate from one area to another through an extracellular matrix. Cell invasion is exhibited by both
normal cells as well as cancerous cells in response to specific external signals, including chemical & mechanical stimuli. During invasion,
extracellular matrix is enzymatically degraded by cellular proteases before cells migrate to the new location. Cell invasion is required for
normal processes such as wound repair, vasculature formation and the inflammatory response as well as the abnormal invasion of tissues
by tumor cells during metastasis. BioVision’s Cell Invasion Assay Kit utilizes a Boyden chamber coated with Collagen I, where the cells
invade the matrix and then migrate through a semipermeable membrane in the Boyden chamber in response to stimulants or inhibitory
compounds. The percent cell invasion can be analyzed directly in a plate reader. Our assay is easy to use, sensitive and adaptable to
high-throughput systems.
II.
Applications:
 Measure cell invasion in response to stimuli
 Screen and characterize compounds that influence cell invasion
III.
Sample Type:
 Invasive cell lines
 Invasion inhibitor or stimuli
IV.
Kit Contents:
Components
K916-100
Cap Code
Part Number
Wash Buffer
Cell Dissociation Solution
Control Invasion Inducer
Cell Dye
Cell Invasion Chamber
Collagen I
2 x 100 ml
10 ml
1.5 ml
1 Vial
1 each
5 ml
NM
NM
Red
Green
Plate
Clear
K916-100-1
K916-100-2
K916-100-3
K916-100-4
K916-100-5
K916-100-6
V.
User Supplied Reagents & Equipment:
 Fluorescence Plate Reader
 Cell Culture Media
 Cotton Swabs
 Centrifuge to spin 96-well plate
 96-well clear bottom white plate
 1 M Tris, pH-7.5 (Cat. # 1075)
VI.
Storage and Reagents Preparation:
Store kit at -20°C, protected from light. Briefly centrifuge small vials prior to opening. Assay is performed under sterile conditions. Read
entire protocol before performing the assay.
 Wash Buffer, Cell Dissociation Solution and Control Invasion Inducer: Store at -20°C. Bring to 37°C before use. Stable for six
months.
 Cell Dye: Add 100 µl of DMSO (Not provided) to the vial. Aliquot and store at -20°C.
 Cell Invasion Chamber: Open under sterile conditions. Keep at room temperature.
 Collagen I: Aliquot under hood and store at 4°C, if needed.
VII.
Cell Invasion Assay Protocol:
1. Add 50 µl Collagen I to coat desired wells of the Top Chamber. Incubate plate at room temperature for 2-3 hrs in flow hood or overnight
2-8°C to form a thin film of Collagen I. Check the chamber from the side to make sure the plates are dried. Incubate for a longer
duration if needed. Wash the coated plate three times with 100 µl 1 M Tris (not provided). Aspirate.
2. Grow enough cells to perform a Cell Invasion Assay and a Standard Curve in desired media and culture conditions. Adherent cells
should be cultured to ~80% confluence.
3. Prior to the assay, starve cells for 18-24 hrs in a serum-free media (0.5% serum can be used, if needed). After starvation, harvest cells
and centrifuge at 1,000 x g, for 5 min. to pellet them. Resuspend the cell pellet in Wash Buffer and count the number of cells using
6
hemocytometer or automated cell counter. Resuspend cells at 1 x 10 cells/ml in a serum-free media.
4. Under sterile conditions, disassemble the Cell Invasion Chamber and carefully remove the plate cover and the top chamber (Fig. a).
Bottom Chamber: Add 200 µl of medium per well containing desired chemoattractant to the bottom chamber. In control well(s), we
recommend omitting the chemoattractant. For Positive Control, add 20 µl of Control Invasion Inducer to 180 µl of medium in the bottom
chamber. Reassemble the top and bottom chambers while ensuring no air bubbles are trapped between them.
Top Chamber: Add 50 µl (~50,000 cells) of cell suspension to each well of the top chamber. Add desired stimulator or inhibitor to the
top well, and gently mix. Make up the volume to 100 µl with media. Carefully replace the plate cover and incubate the Cell Invasion
Chamber at 37°C in CO2 incubator for 2-48 hrs.
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected]
FOR RESEARCH USE ONLY!
Note: Invasive cells pass through the Collagen I membrane and cling to the outer side of the top chamber. Non-invasive cells stay in
the upper chamber.
5. Standard Curve:
A. Each cell type requires a separate Standard Curve. Prepare a Standard Curve by adding 50 µl cell suspension (1 x 106 cells/ml, ~
50,000 cells) in desired well(s) in a 96-well plate (white plate clear bottom). Serially dilute the cells 1:1 in Wash Buffer and generate
a Standard Curve of cells (50,000, 25,000, 12,500, 6,250, 3,125, 1,562, 781, and 390) in 100 µl total volume. As blank, use 100 µl
of Wash Buffer.
B. Dilute Cell Dye 1:250 in PBS and add 50 µl of diluted Cell Dye to each well. Incubate at 37°C for 30 min. Read the fluorescence at
Ex/Em = 485/530 nm. Plot the Standard Curve (Number of Cells Vs RFU obtained). Fit the data points using a linear trend line with
zero intercept. The equation for the straight line and R-squared value are used for data analysis of samples.
Note: The Cell Invasion RFU reading should fall in the linear range of the Standard Curve. We recommend using triplicates for
Standard Curve.
6. Separation of Invasive Cells:
A. After the desired incubation with cell invasion inducers/inhibitors, carefully remove the plate cover and aspirate media from the top
chamber without puncturing the membrane and matrix.
B. Remove cells from the top chamber using a cotton swab. Disassemble the Cell Invasion Chamber by removing the top chamber.
Invert the top chamber and set it aside.
C. Place the plate cover on top of bottom chamber and centrifuge the plate at 1,000 x g for 5 min. at room temperature.
D. Carefully aspirate the media from the bottom chamber, and wash the chamber with 200 µl Wash Buffer.
E. Centrifuge the plate at 1,000 x g for 5 min. at room temperature and aspirate the Wash Buffer from the bottom chamber.
7. Count Invasive Cells:
A. Dilute Cell Dye 1:500 in Cell Dissociation Solution. Mix well. Make desired amount of Cell Dye solution depending on the number of
wells.
B. Add 100 µl of the mix to each well of the bottom chamber. Reassemble the Cell Invasion Chamber by placing the top chamber into
the bottom chamber. Incubate at 37°C in CO2 incubator for 30 min*. After incubation, disassemble the Cell Invasion Chamber and
remove the top chamber.
C. Read the plate at Ex/Em = 485/530 nm. Calculate the number of cells invaded using the equation of the straight line obtained from
Standard Curve. Percentage Invasion can be calculated as follows:
# 𝐂𝐞𝐥𝐥𝐬 𝐢𝐧 𝐋𝐨𝐰𝐞𝐫 𝐂𝐡𝐚𝐦𝐛𝐞𝐫
% 𝐈𝐧𝐯𝐚𝐬𝐢𝐨𝐧 = (
) 𝒙 𝟏𝟎𝟎
𝐓𝐨𝐭𝐚𝐥 # 𝐂𝐞𝐥𝐥𝐬 𝐚𝐝𝐝𝐞𝐝 𝐭𝐨 𝐓𝐨𝐩 𝐂𝐡𝐚𝐦𝐛𝐞𝐫
*Note: During incubation with Cell Dissociation Solution/Cell Dye, gently tap the plate on the side to ensure optimal dissociation of
the invasive cells that cling to the outer side of the top chamber.
a.
b.
c.
120
% Invasion
100
RFUs
80
60
40
20
y = 0.0089x + 6.0026
30
25
20
15
10
5
0
0
0
5,000
10,000
Cell Number
-
+
-
+
Non Invasive
Invasive
BALB/3T3
HT-1080
Figure 1: (a) Cell Invasion plate: The cells are added to the Top Chamber and the Control Invasion Inducer or chemoattractant
are added to the Bottom Chamber. (b) Standard Curve: HT-1080 cells were harvested, counted and serially diluted to obtain
desired cell number. Cells were incubated according to the protocol. (c) Cell Invasion: NIH-3T3 and HT-1080 cells were starved
overnight and treated with Control (Cnt) Invasion Inducer or remain untreated (No Treatment). Treatment with Control Invasion
Inducer demonstrated a significant increase in invasion of HT 1080 cells as compared to NIH-3T3 control cells.
VIII. RELATED PRODUCTS:
EZCellTM Invasion Assay (Basement Membrane), 96-well, 8 µm (K912)
EZCellTM Invasion Assay (Laminin), 24-well, 8 µm (K915)
EZCellTM Invasion Assay (Collagen I), 96 and 24-well, 8 µm (K916, K917)
EZViable™ Calcein AM Cell Viability Assay Kit (K305)
EZCellTM Invasion Assay (Collagen IV), 96-well, 8 µm (K918)
EZCellTM Invasion Assay (Collagen IV), 24-well, 8 µm (K919)
FOR RESEARCH USE ONLY! Not to be used on humans
155 S. Milpitas Blvd., Milpitas, CA 95035 USA | T: (408)493-1800 F: (408)493-1801 | www.biovision.com | [email protected]